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EP2814985A2 - Procédé extrêmement sensible de détection de mutations à faible fréquence - Google Patents

Procédé extrêmement sensible de détection de mutations à faible fréquence

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Publication number
EP2814985A2
EP2814985A2 EP20130749733 EP13749733A EP2814985A2 EP 2814985 A2 EP2814985 A2 EP 2814985A2 EP 20130749733 EP20130749733 EP 20130749733 EP 13749733 A EP13749733 A EP 13749733A EP 2814985 A2 EP2814985 A2 EP 2814985A2
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European Patent Office
Prior art keywords
blocker
wild type
edge
pcr
nucleic acid
Prior art date
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Withdrawn
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EP20130749733
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German (de)
English (en)
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EP2814985A4 (fr
Inventor
Haiying Wang
Yuqiu Jiang
Yixin Wang
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Janssen Diagnostics LLC
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Janssen Diagnostics LLC
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Publication of EP2814985A2 publication Critical patent/EP2814985A2/fr
Publication of EP2814985A4 publication Critical patent/EP2814985A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • a patient may harbor cells that have one or more single point substitution, insertion, or deletion mutations that play a deleterious role. Different patients may have different mutations even when having the same 'type' of cancer. Each mutation or group of mutations may be best treated by different therapeutic regimes. But, such point mutations are often far more difficult to detect than large insertions or deletions or germ line mutations since they are camouflaged by normal tissue which yields a large signal due to the identity of virtually all of nucleic acid sequence flanking the mutation site with genetic material obtained from surrounding normal tissue in a sample.
  • PCR Polymerase Chain Reaction
  • a PCR based amplification may simply not readily amplify the nucleic acid variant present at a low frequency— typically present in a tumor cell since the variant is often present at a much lower level than normal tissue in a sample (often at just a fraction of a percent)— when attempting early detection of a cancer or detection and monitoring by relatively non-invasive means.
  • AS-RT-PCR Allele-Specific amplification using Real-Time Polymerase Chain Reaction technology
  • This technique helps evaluate genetic mutations present at a low frequency in a patient— such as in tumor cells or in a chimera. This technique can help detect genetic variants, single nucleotide polymorphisms (SN P) and genetic mutations present at low frequency— as is demonstrated herein later.
  • the Morlan reference describes the use of center-blocker oligonucletides to enhance the detection of point mutations— which is an improvement of the allele specific amplification technique reported by Wu et al. in "Allele-specific enzymatic amplification of f8- globin genomic DNA for diagnosis of sickle cell anemia", Proc. Natl. Acad. Sci. USA 86:2757-60 (1989). I n the Morlan technique a mutant specific primer is used together with a center-blocker oligonucleotide. By center-blocker
  • oligonucleotide is meant an oligonucleotide with a mismatch that is about equidistant from either end.
  • the mutant specific primer is entirely
  • the mutant specific primer of Morlan is further trimmed at its 5' end to reduce its melting temperature to about 10°C below the anneal/extend temperature used in the PCR.
  • the center blocker oligonucleotide is complementary to the wild type sequence and spans the site of a point mutation so that the point mutation is about equally spanned by it.
  • the center blocker oligonucleotide is further phosphorylated at its 3' end to prevent extension during a PCR reaction.
  • AS-NEPB-PCR Non-Extendable Primer Blocker Allele Specific-Real Time Polymerase Chain Reaction
  • the disclosed method enables a universal design of Allele Specific primer and primer blocker that can be used in any of AS-RT-PCR assays to detect SNP or genetic mutations. The method simplifies assay optimization procedures and achieved 0.1% detection sensitivity with close to 100% specificity.
  • the disclosed edge-blocker oligonucleotide based AS-NEPB-PCR method amplifies allele specific DNA (or RNA) while dramatically blocking amplification of wild type (WT) DNA (or RNA).
  • the disclosed AS-NEPB-PCR design allows ready modification of an existing PCR reaction setup by introducing an edge-blocker oligonucleotide together with an allele specific primer complementary to the mutant sequence to achieve allele specific amplification.
  • the edge-blocker oligonucleotide and allele specific primer may have the same length and differ only at the 3'end where the edge-blocker oligonucleotide has a non-complementary base relative to the mutant sequence and a blocked 3'end while the allele specific primer is preferably entirely complementary to the mutant sequence of interest and has a hydroxyl group at its 3' end to allow extension during a PCR reaction.
  • This method is not only simpler to implement but also successful in routinely suppressing the edge-blocker oligonucleotide and allele specific primer.
  • edge-blocker oligonucleotide based AS-NEPB-PCR method is not limited to just detecting point mutations, but can detect specified insertions or deletions as well.
  • the edge-blocker oligonucleotide based AS-NEPB-PCR method was used to detect three different genetic mutations in cancers.
  • the genetic mutations targeted here were in KRAS, BRAF, and EGFR genes, which were detected with the use of three different types of modified edge-blocker oligonucleotides (phosphate, inverted dT and amino-C7).
  • the resulting data were compared to one of the known common blocking methods as a reference.
  • the novel method disclosed herein was able to detect one copy of mutant DNA in 1000-copy of normal DNA background of a heterogeneous sample, and was far more sensitive than the reference blocking method.
  • a preferred method for detecting a mutant nucleic acid sequence, defined by one or more mutations due to at least one or more of a substitution, a deletion or an insertion, while suppressing the signal due to the wild type sequence includes several steps.
  • a primer complementary to the mutant nucleic acid sequence is selected such that its 3' end matches up with at least one mutated nucleic acid position.
  • a second primer, an edge-blocker wild type oligonucleotide is also used.
  • the edge-blocker wild type oligonucleotide corresponds to the wild type sequence such that the 3' end of the edge-blocker wild type primer has at least one mismatch at or about its 3' end relative to the mutant nucleic acid sequence but has no mismatches relative to the wild type sequence.
  • the 3' hydroxyl group at end of the edge-blocker wild type primer is blocked whereby making it non-extendable in a polymerase chain reaction.
  • reverse primers are selected as usual although it should be noted that when trying to detect deletions or insertions, it may be advantageous to use reverse primers similar to the one corresponding to the wild type sequence having a blocked 3' end.
  • the amplification products of a PCR reaction are detected with at least one probe specific for the amplified product in a polymerase chain reaction.
  • the polymerase chain reaction is a real-time polymerase chain reaction.
  • one or more probes may be added after the polymerase chain reaction is initiated. Further, the initial starting materials may be generated using a reverse transcriptase to investigate transcription products for point or other mutations of interest.
  • this method can use the 3' end of the edge-blocker wild type oligonucleotide with at least one mismatch at or about its 3' end relative to the mutant nucleic acid sequence— even two mismatches to cover both the point mutations to even more effectively suppress the amplification of the wild type sequence. It is preferred that the allele specific primer cover both the mutant positions. With such coverage even if there is extension based on the binding of the allele specific primer, the amplification products will correspond to the target mutations rather than the wild type sequence.
  • edge-blocker wild type oligonucleotide may have a mismatch, but may be counterbalanced by increasing the length of the edge-blocker wild type oligonucleotide to suppress amplification of the wild type sequence by the allele specific primer.
  • the edge-blocker wild type oligonucleotide is equal in length to the allele specific primer with both having 3' ends that cover similar portions of the mutant or wild type sequence.
  • the method can detect a mutant sequence even when it is present at a level of only about 1 in 1000 or even rarer.
  • edge-blocker wild type oligonucleotide may be longer at its 5' end than the allele specific primer to assist it in competing out the allele specific primer to prevent accidental extension of the wild-type sequence by the allele specific primer.
  • the melting temperature of the allele specific primer is lower than that for the edge-blocker wild type oligonucleotide relative to the wild type sequence.
  • the melting temperature of the allele specific primer is lower, e.g., about 10°C lower, than that for the edge-blocker wild type oligonucleotide.
  • edge-blocker wild type oligonucleotide Effective competition by the edge-blocker wild type oligonucleotide for the wild type sequence is helped by ensuring that the edge-blocker wild type oligonucleotide is present at a concentration suitable for suppression of the wild- type sequence while allowing amplification of the mutant sequence.
  • this concentration is comparable— while being at least equal— to the level of the wild type sequence concentration.
  • the concentration of the allele specific primer is in excess of that of the wild type sequence since it is incorporated into the PCR product while the edge-blocker wild type oligonucleotide serves to suppress amplification of the wild-type sequence, the likelihood of which decreases as the allele specific primer levels decrease with amplification of the target PCR product.
  • the concentrations of the edge-blocker wild type oligonucleotide and the allele specific primer are comparable.
  • the disclosed method for the detection of rare mutant nucleic acid sequence includes quantitation to estimate a level of the mutant nucleic acid sequence relative to the wild type sequence.
  • a calibration curve may be generated by spiking the samples for a polymerase chain reaction.
  • the disclosed method is a diagnostic method suitable for early detection of cancer by way of detecting the presence of one or more target cells in a sample derived from a patient, which cells harbor a mutant nucleic acid sequence, and the presence of which cells likely leads to malignancy or recurrence.
  • the method comprises selecting an allele specific primer
  • an edge- blocker wild type oligonucleotide corresponding to the wild type sequence such that the 3' end of the edge-blocker wild type oligonucleotide has at least one mismatch at or about its 3' end, and, wherein, furthermore, the 3' end of the edge-blocker wild type oligonucleotide is blocked whereby making it non- extendable competitive inhibitor in a polymerase chain reaction.
  • a polymerase chain reaction preferably a real time polymerase chain reaction is carried out.
  • the method not only detects cancer usefully early, but also can guide one to therapies best suited for treating the patient.
  • the mutant nucleic acid sequence is detected by detecting the reaction products less than a pre-specified number of amplification cycles.
  • mutant nucleic acid sequence's presence is detected if amplification products corresponding to it are detected but a reference sequence, treated like the mutant sequence is not detected in the same sample.
  • the target sequence with or without its corresponding wild-type like sequence, may be used to spike the sample. This can determine sensitivity and other parameters of interest.
  • edge-blocker wild type oligonucleotide is blocked by derivatizing or replacing its 3' hydroxyl group with one or more selected from the group consisting of phosphate, inverted dT and amino-C7.
  • Figure 1 General diagram for edge-blocker oligonucleotide based AS-N EPB-PCR design: Primer with 3' end modification (phosphate or inverted dT) functions as a blocking group to prevent polymerase extension on wild type sequence. - PCR amplifies only AS primed mutation while WT strain is blocked by a modified non- extendable primer (edge-blocker oligonucleotide based AS-N EPB-PCR).
  • Edge-blocker oligonucleotide based AS-NEPB-PCR WT-1 and 2 stand for edge- blocker oligonucleotide-1 and edge-blocker oligonucleotide-2 and they correspond to Seq Id. 5 and Seq. Id. 6 respectively.
  • Probe-dye (Dye labeled probe— Seq. Id. 4) and reverse primer (Seq. Id. 3) are common for both assays.
  • AS primer-1, Seq. Id. 1 runs with edge-blocker oligonucleotide-WT-1 (NEPB-WT- 1), Seq. Id. 5, and AS primer-2, Seq. Id. 2, with edge-blocker oligonucleotide-WT-
  • FIG. 3 Mapping of edge-blocker oligonucleotide designs for two K-ras gene mutations.
  • AS Primer-KrasP4 (Seq. Id. 17) and KrasP7 (Seq. Id. 21) indicate G12V- AS-Forward Primer and G13D-AS-Forward Primer.
  • NEPB-WT-KrasP4B (Seq. Id. 18) and P7B (Seq. Id. 22) stand for G12V-NE edge-blocker oligonucleotide and G13D-N E edge-blocker oligonucleotide.
  • KProbel (Seq. Id. 20) and KProbe2 (Seq. Id.
  • FIG. 4 BRAF V600E mutant detection: 8 out of 20ul PCR products from the 20ng DNA reaction were loaded on the gel. The single sharp bands were observed from 5% to 0.1% mutant reactions and no PCR products were observed from SKBR3 WT AS-NEPB2-PCR-2 reaction, except Actin-PCR products.
  • Figure 5a KrasP4 (G12V; G>T) NEPB or CBO blocker PCR on SW480 Cell line DNA: 8 out of 20ul PCR products from the 20ng DNA reaction were loaded on the gel. Clean PCR products were visualized on a 4% agarose gel from 0.1% mutant reactions and no PCR products were observed from WT reaction, except Actin- PCR products. The PCR product was also visualized from WT amplification without adding any Blockers. All of NTC was not undetermined.
  • FIG. 5b KrasP7 (G13D; 13G>A) N EPB or CBO blocker PCR on HCT116 Cell line DNA: 8ul PCR products were loaded on the gel. Clean PCR products were visualized from 0.1% mutant reactions and no PCR products were observed from WT reaction. The PCR product was visualized as well from WT amplification without adding any Blockers.
  • Figure 6. EGFR Exon 21 L858R (2573 T>G) AS-NEPB-PCR on NCI-H1975 Cell line DNA: 8ul PCR products were loaded on the gel. Clean PCR products were visualized from 0.1% mutant reactions and no PCR products were observed from WT reaction. The PCR product was visualized as well from WT amplification without adding any Blockers.
  • AS-N EPB-PCR Non-Extendable Primer Blocker Allele Specific-Real Time Polymerase Chain Reaction
  • HT29 cell line (ATCC# HTB-38D) is heterozygous and SK-M EL28 cell line (ATCC# CRL-5908) is homozygous in BRAF mutation with predicted mutation effect of p.V600E (c.l799T>A). Characterization of BRAF mutation was described as likely oncogenic mutation (11).
  • HCT 116 cell line (ATCC# CCL-247) has a mutation in codon 13 (p.G13D; c.G>A) and SW480 cell line (ATCC# CCL-228) has a mutation in codon 12 (p.G12V; c.G>T) of K-ras protooncogene.
  • NCI-H1975 cell line (ATCC# CRL-5908) carries EGFR Exon 21 recurrent heterozygous missense mutation of L858R-2573T>G (12).
  • SKBR3 cell line (ATCC# HTB-30) was used as wild type control for BRAF, K-ras and NCI-H358 cell line (ATCC# CRL-5807) as wild type control for EGFR mutation detection assays.
  • Melanoma and Colon tissue samples were purchased from ProteoGenex (Culver City, CA. US), one of the providers of biological specimens.
  • CTC circulating tumor cells
  • Cell line DNA was extracted by using AllPrepTM DNA/RNA Micro Kit and FFPE tissue DNA was extracted by using RNeasy FFPE kit from Qiagen (Valencia CA. US Cat# 80284 and 74404) according to the manufacturer's instructions. Then, extracted DNA was quantified on Nanodrop-2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE. US) following the User Manual and stored at -20°C until later use.
  • NEPB A general diagram for NEPB is demonstrated in Figure 1.
  • allele-specific primers can be designed to either positive or negative DNA (or RNA) strand. Either for the forward or the reverse primer, 3'- end is anchored on the variant base.
  • the melting temperatures (Tm) of ASP should be close to the PCR extension temperature.
  • Edge-blocker oligonucleotide based AS-NEPB-PCR method was designed to the same strand and length as the allele-specific primer except the forward or reverse primer 3'-end is anchored on the WT base and not extendable by polymerases with 3' end modification (phosphate or inverted dT or amino-C7).
  • Center-blocker oligonucleotide design was based on the criteria listed in the paper (7) and used for comparison to edge-blocker oligonucleotide based AS- NEPB-PCR method. The design sequences were assembled by SeqMan II expert sequence analysis software (DNASTAR Inc, Wl, US); Figure 2 for the design of BRAF gene and Figure 3 for two K-ras genes.
  • oligonucleotides including primers, probe and blocker, were purchased from Biosearch Technologies, Inc (Novato, CA. US), except MGB probes. Modified oligonucleotides including Fluorophore dye (FAM and CAL Fluor Orange) labeled at 5' ends and BHQ or Phosphate at 3 'ends (Table la, b and C) were synthesized according to the manufacturer's instructions. Two MGB probes for K-ras assay were purchased from Applied Biosystems (Foster City, CA). AS-NEPB- PCR Amplification
  • the AS-NEPB-PCR assay for allele analysis of B-Raf, K-ras and EGFR included one ASP on the positive strand, one NEPB, one fluorescence labeled sequence specific TaqMan probe with BHQ or MGB at 3'end and one non-AS reverse primer (RP) on the negative strand.
  • the sequences of primers, probe and oligonucleotide blockers are listed in Tablela, Talelb and Table lc below for the BRAF gene, Kras gene and EGFR gene respectively. All Tables are provided in the section titled 'Tables', which follows the 'References' section.
  • the assay was run as singlex or duplex AS- RT-PCR format, AS gene with Internal Control gene in two individual reactions or in one reaction, on Applied Biosystems 7500 (or 7900) Real-Time PCR System (Foster City, CA).
  • each primer, blocker and probe of AS-NEPB-PCR assay For BRAF p.V600E (c.l799T>A) detection, the final concentrations of each primer, blocker and probe of AS-NEPB-PCR assay are listed in Tables 2a.
  • the assay was set up as follows: lOng to 50ng of DNA heterogeneous mixture was used and was carried out in a final volume of 20 ul in reaction.
  • the AS-NEPB-PCR was carried out using TaqMan ® Gene Expression Master Kit (Applied Biosystems, Part # 4368814). Each reaction consisted of 10.0 ul of 2X PCR Master Mix, 1 ul of 20x primer/blocker/probe mix, and 1-5 ul of 10 ng /ul total DNA sample.
  • the AS- NEPB-PCR assays were run as follows: 1 cycle of denaturation at 95 °C for 10 min, 40 cycles of 95 °C for 20 seconds denaturation and 64°C in favor of BRAF-NEPB1- PCR-1 or 58 °C in favor of BRAF-NEPB2-PCR-2 for 45 seconds annealing and extension under run Standard Mode.
  • each primer, blocker oligonucleotides and probes of AS-NEPB-PCR assay is listed in Tables lb and 2b.
  • the assay was set up as follows: 20ng of DNA heterogeneous mixture was used and was carried out in a final volume of 20 ul in reaction.
  • the AS-NEPB-PCR was carried out using TaqMan ® Gene Expression Master Kit. Each reaction consisted of 10.0 ul of 2X PCR Master Mix, 2 ul of lOx primer/blocker/probe mix, and 2 ul of 10 ng /ul total DNA sample.
  • the AS-NEPB-PCR assays were run as follows: 1 cycle of denaturation at 95 °C for 10 min, 40 cycles of 95 °C for 20 seconds denaturation and 60°C for 45 seconds annealing and extension under run Standard Mode.
  • center-blocker oligonucleotide based AS -PCR method for K-ras was run at the same PCR condition as the edge-blocker oligonucleotide based AS- NEPB-PCR method except for using 4x center-blocker oligonucleotide concentration as corresponding ASP concentration, which was suggested in the publication (7).
  • each primer, blocker oligonucleotides and probes of AS-NEPB-PCR assay is listed in Tables lc and 2c.
  • the assay set-up was the same as K-ras mutation assay except DNA template.
  • DNA samples were from NCI-H1975 and NCI-H358 heterogeneous mixture.
  • the AS-NEPB-PCR assays were run as follows: 1 cycle of denaturation at 95 °C for 10 min, 40 cycles of 95 °C for 20 seconds denaturation and 63°C for 45 seconds annealing and extension under run Standard Mode.
  • Edge-blocker oligonucleotide based AS-NEPB-PCR detection sensitivity/specificity of BRAF (V600E) and K-ras (G12V or G13D) were estimated by using dilutions of the related mutant cell line DNA (describe the above Cell Line Sample section) in wild-type DNA of the cell lines SKBR3. Dilutions were made at 5%, 1%, 0.5% and 0.1% mutant DNA and data were collected and analyzed by ABI 7500 fast System SDS software (Applied Biosystems). The same analysis method was used for both center-blocker oligonucleotide based AS- NEPB-PCR and edge-blocker oligonucleotide based AS-NEPB-PCR methods.
  • PCR aliquots were also analyzed by agrose gel electrophoresis with 100 bases molecular marker (Invitrogen, Carlsbad, CA). One specific PCR product from a corresponding positive sample should be present after amplification.
  • center-blocker Oligo (CBO) method was first adapted from the publication of K-ras mutation detection (7), the ASP and blocker designs were followed the criteria listed in the paper.
  • Assay conditions were tested in order to reach 0.1% detection sensitivity of BRAF mutation gene.
  • We have tried to optimize assay conditions by titrated various annealing temperature (58, 60, 62, 64 and 65°C) and ratio of ASP: PB (1:4, 1:2 and 1:1).
  • annealing temperature 58, 60, 62, 64 and 65°C
  • ratio of ASP: PB (1:4, 1:2 and 1:1.
  • none of conditions could reach 0.1% mutant detection sensitivity and without non- specific amplification on WT template.
  • Edge-blocker oligonucleotide (EBO) based AS-NEPB-PCR method was developed to improve detection sensitivity and remove non-specific amplification for BRAF gene mutation detection assay.
  • EBO Edge-blocker oligonucleotide
  • a common reverse primer and probe were designed downstream of the polymorphic site and used in AS-N EPB-PCR.
  • edge-blocker oligonucleotide based AS-NEPB-PCR enhanced the sensitivity of the AS-PCR, without non-specific amplification on WT DNA. It performed better than CBO method (Table 3).
  • Edge-blocker oligonucleotide based AS-N EPB-PCR method also showed strong allele specific amplifications, detected one copy of mutant DNA in 1000-copy normal DNA background of heterogeneous mixture (0.1% mutation frequency and 2-3 mutant copies) in both AS-NEPB-PCR assays.
  • NEPB2-PCR-2 in both cell line and FFPE tissue DNA samples.
  • the 100% sensitivity and specificity was obtained by using AS-NEPB-PCR method based on the sequence data (Table 5).
  • edge-blocker oligonucleotide based AS-N EPB-PCR method was also verified on two KRAS gene mutants (p.G12V; G>T and p.G13D; 13G>A) and compared to the center-blocker oligonucleotide based AS-PCR method. A small number of ASP vs. edge-blocker oligonucleotide ratios were tested to obtain the best concentration of edge-blocker oligonucleotides. The same AS primers described in the paper were used under the annealing temperature 60°C suggested by the paper (7).
  • Edge-blocker oligonucleotides modified by inverted dT or amino-C7 were also evaluated.
  • the equivalent assay performances were obtained as 3' end modified by Phosphate (Table 8).
  • edge-blocker oligonucleotide based AS-N EPB-PCR method was tested on 42 clinical samples, circulating colorectal tumor cells.
  • BRAF (V600E) mutations were detected in two tissue samples and one CTC sample, which were matched with the sequencing data. Non-specific amplification was not observed in both tissue and CTC samples which confirmed by sequencing data (Table 9).
  • Edge-blocker oligonucleotide based AS-N EPB-PCR method was also evaluated on EGFR gene (exon 21_L858R) mutation detection. The results showed 0.1% of mutations ( ⁇ 5 copies) were detected without non-specific amplification at 1:1 ratio of ASP to edge-blocker oligonucleotide and Annealing Temp 63°C (Table 10 and Figure 6). Good assay precision, ⁇ 2% CV, was obtained from 5%, 1% and 0.1% in the triplicates.
  • Edge-blocker oligonucleotide based AS-NEPB-PCR method has been employed on the detection of 3 different genes (B-Raf, K-Ras, and EGFR) and 4 mutants (V600E, G12V, G13D and L858R) effectively.
  • Optimal assay conditions were determined easily for each of the assays due to the advantage of edge- blocker oligonucleotide design, which has the same strand and length as the allele-specific primer producing almost the same melting temperatures (Tm) as ASP.
  • Tm melting temperatures
  • edge-blocker oligonucleotide based AS-N EPB-PCR method is a highly sensitive and specific method for mutation detection in highly heterogeneous samples. Also, the edge-blocker oligonucleotide based AS-NEPB- PCR method provides great advantages in simplifying assay design and assay optimization over the other blocking method. Edge-blocker oligonucleotide based AS-NEPB-PCR method allows an efficient workflow when a number of different mutation assays need to be developed.
  • Tm melting temperatures
  • Table 1C Primer/Probe/blocker Sequences and Labeling for EGFR gene (L858R;
  • Table 3 lOng DNA of HT29 mutant/SKBR3 WT mixtures at various ratios were input into the PCR reactions for BRAF V600E mutant detection, 0.1% of mixtures equivalent to ⁇ 3 copies of mutant. Undetermined in PCR was considered as C T 40 during delta C T calculations. Both NE Primer blocker, EBO, method reached 0.1% detection sensitivity without non-specific amplification. NE Primer blocker- 2 showed the best results to discriminate alleles (bigger delta C T between WT and mutant) compared to CBO blocker method. CBO blocker-1 showed 0.5% detection sensitivity and non-specific amplification on WT DNA. However, the delta C T between WT and mutant was ⁇ 3Ct less than NEPB method. CBO blocker- 2 gave constantly non-specific amplification if having 0.1% detection sensitivity. All the final reaction conditions were described in the Method of "AS-NEPB-PCR Amplification".
  • Table 4 10-50ng DNA of HT29 mutant/SKBFG WT mixtures at various ratios were input into AS-NEPB2-PCR-2 of BRAF V600E mutant detection. The method detected constantly 0.1% of mutation rate without non-specific amplification. All of Actin C T s were ⁇ 25 and NTC was not undetermined.
  • Table 5 The assay gave consistent performance on detecting the mutated allele without non-specific amplification of DNA samples, which were confirmed by sequencing. NVD-No variants detected.
  • NVD Table 6a KrasP4 (G12V; G>T) AS-NEPB-PCR on SW480 Cell line DNA: 20ng DNA of SW480 mutant/SKBR3 WT mixtures at three ASP:NEPB ratios were input into the PCR reactions, 0.1% of mixtures equivalent to ⁇ 5 copies of mutant. PCR conditions were as stated previously. The ratio of 1:1 gave the best result that reached 0.1% detection sensitivity without non-specific amplification. All of NTC was not undetermined.
  • N EPB a nd CBO Blocker methods were tested on SW480 and HCT116 Cell line DNA with ASP of KrasP4 and KrasP7: 20ng DNA of each mutant/SKBFG WT mixtures with 1: 1 ratio of ASP: N EPB or 1:4 ratio of ASP: CBO blocker was used the PCR reactions. PCR conditions were as stated previously. N EPB method showed the equivalent, if not better, assay performances as CBO Blocker method; reached 0.1% detection sensitivity without non-specific am plification on WT DNA. All of NTC was not undetermined.
  • Table 8 20ng DNA of HT29 mutant (BRAF V600E)/SKBR3 WT mixtures at various ratios were input into AS-NEPB-PCR. Three N EPB modifications, Phosphate or inverted dT or amino-C7, gave equivalent results. The method detected 0.1% of mutation rate without non-specific amplification. All of Actin Cts were ⁇ 25 and NTC was not undetermined.
  • BRAF (V600E) mutations were detected in the two clinical tissue samples (2/42) and one CTC sample (1/42), which were 100% (tissues) or 50% (CTCs), matched with sequencing data.
  • CTC samples the mutation only was detected for CTC-196 which had 5 CTCs, but not detected for CTC-220 which had no CTC account based on the analysis of CellSearch system.
  • Non-specific amplification was not observed in both tissue and CTC samples which were confirmed by their sequence data. All of Actin C T s were ⁇ 25 (data not shown).

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Abstract

L'invention concerne un procédé AS-NEPB-PCR en fonction d'un oligonucléotide bloqueur de bord qui amplifie l'ADN (ou l'ARN) spécifique d'un allèle tout en bloquant significativement l'amplification de l'ADN (ou l'ARN) de type sauvage (WT). La conception AS-NEPB-PCR permet une modification facile d'un réglage de réaction de PCR existant par un oligonucléotide bloqueur de bord conjointement à une amorce spécifique d'un allèle complémentaire à la séquence mutante pour obtenir une amplification spécifique de l'allèle. Le procédé simplifie des processus d'optimisation de dosage et permet une sensibilité suffisante pour détecter un signal présent à un niveau de 0,1 % et proche d'une spécificité de 100 %, ce qui est utile dans la détection de SNP ou de mutations génétiques. Le procédé a été utilisé pour détecter trois mutations génétiques différentes dans le cancer, dans KRAS, BRAF et EGFR, avec trois différents types d'oligonucléotides modifiés, bloqueurs de bord (phosphate, dT inversé et amino-C7). Il a été possible de détecter une copie de l'ADN mutant dans 1000 copies d'un fond normal d'ADN d'un échantillon hétérogène, et ceci s'est révélé être beaucoup plus sensible que l'autre procédé de blocage.
EP13749733.5A 2012-02-15 2013-02-13 Procédé extrêmement sensible de détection de mutations à faible fréquence Withdrawn EP2814985A4 (fr)

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JP (1) JP2015517798A (fr)
AU (1) AU2013221642A1 (fr)
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WO (1) WO2013123031A2 (fr)

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JP2015517798A (ja) 2015-06-25
IL234021A0 (en) 2014-09-30
US20160130641A1 (en) 2016-05-12
WO2013123031A3 (fr) 2015-03-12
WO2013123031A2 (fr) 2013-08-22
EP2814985A4 (fr) 2016-03-30
AU2013221642A1 (en) 2014-09-25

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